Project Summary We have identified endomucin (EMCN), a component of the endothelial cell (EC) glycocalyx, to be a novel regulator of VEGFR2 signaling. siRNA-mediated knockdown of EMCN in human retinal capillary EC blocks VEGF-induced angiogenic functions (proliferation, migration, and tube formation) in vitro and neovascularization in vivo. Our data indicate that EMCN is necessary for VEGF-stimulated VEGFR2 internalization. We hypothesize that EMCN regulates VEGF-induced VEGFR2 endocytosis, and thus VEGF signaling in EC. We propose: (i) To use a genetic approach to examine the role of EMCN in developmental angiogenesis and pathologic neovascularization as well as in adult vascular stability. We have generated mice with floxed EMCN that will be bred with Rosa-Cre to assess the effect of total EMCN knockout, and with tamoxifen-inducible VE-cadherin to examine EC-specific knockout. (ii) To elucidate the molecular mechanism through which EMCN regulates VEGFR2 internalization and signaling, cell biological and biochemical methods will be employed to determine how EMCN functions in VEGF-VEGFR2 endocytosis, to elucidate the structural characteristics of EMCN necessary for its role in VEGFR2 endocytosis, and to identify EMCN-VEGFR2 binding proteins that may be involved in VEGFR2 internalization. (iii) To develop a monoclonal antibody that interferes with the association between EMCN and VEGFR2, the glycosylated extracellular domain of EMCN will be used as an antigen. Our preliminary data using truncation mutants of EMCN indicate that the extracellular domain of EMCN is necessary for its effect on VEGFR2 signaling. Antisera will be screened on the basis of its effects on VEGF-induced EC migration and VEGFR2 internalization. Antisera, that we have shown to interfere with the effect of EMCN on VEGF-induced migration and VEGFR2 internalization, will be tested for its the ability to block pathologic VEGF-induced permeability and angiogenesis in vivo ? alone, compared to aflibercept (VEGF trap), or as a combination therapy with aflibercept. VEGF neutralization is the primary mode of treatment for a number of ocular pathologies that involve neovascularization and vessel permeability. While remarkably successful, there is a significant proportion of patients who appear unresponsive to anti-VEGF therapy. In addition, a number of non-vascular cells in the retina express VEGFR2, and are thus vulnerable to chronic neutralization of local VEGF, with implications for neurotrophic and survival functions. Results of these studies will provide new information about the role of EMCN in vascular development, vascular integrity, and pathologic vessel growth; will reveal novel insights into the regulation of VEGF-stimulated VEGFR2 signaling; and, will test EMCN as a unique endothelial cell-specific target for blocking abnormal VEGF-induced angiogenesis and vascular permeability. !